18S amplicon metabarcoding of marine metagenome: Gulf of Mexico (USA)
Samples were collected and filtered onto Sterivex 0.22 um cartridge filters. DNA was extracted from Sterivex by adding lysis buffer and magnetic bead-based extraction kits (ZymoBIOMICS 96 DNA/RNA MagBead kit). Following bead beating in the cartridges, extractions were finished on an automated KingFisher Flex instrument (Thermo Fisher) in 96-well plates. A two-step PCR approach was used, targeting 18S V0 rRNA with primers 1391f; 5-GTACACACCGCCCGTC-3 and EukBr; 5-TGATCCTTCTGCAGGTTCACCTAC-3. Primers were constructed with Fluidigm common oligos CS1 forward (CS1-TS-F: 5-ACACTGACGACATGGTTCTACA-3) and CS2 reverse (CS2-TS-R: 5-TACGGTAGCAGAGACTTGGTCT-3) fused to their 5' ends. PCR products were sent to the Michigan State University Research Technology Support Facility Genomics Core for secondary PCR and sequencing. Secondary PCR used dual-indexed, Illumina-compatible primers, targeting the Fluidigm CS1/CS2 oligomers at the ends of the PCR products. Sequencing runs were performed on an Illumina MiSeq to produce 250+250 nt paired reads